1. Field of the Invention
The present invention relates to rate immunonephelometric techniques of analyzing precipitate-forming antigen-antibody reactions and, more particularly, to methods of determining the antigen excess or antibody excess condition of such reactions.
2. Description of the Prior Art
The aforementioned Anderson et al. application discloses a system for the nephelometric assay of antigens and antibodies. For this purpose, an antigen-antibody precipitate forming reaction is conducted in an optically transparent sample container or vial. An excitation system directs a beam of light into the sample container and a detection system measures light scattered at a forward angle by the precipitate. The detected nephelometric or light scatter signal is differentiated to derive a rate signal the peak value of which provides a measure of the concentration of the desired antigen or antibody reaction component.
In immunonephelometric analysis, antigen-antibody reactions are conducted to determine the concentration of sample antigen or the concentration of sample antibody. For antigen determination, the reaction is conducted using antibody as reagent for sample antigen. Conversely, for antibody determination, the reaction is conducted using antigen as reagent for sample antibody. For ease of presentation, the present specification treats only the determination of antigen concentration. However, it should be understood that terms "antigen" and "antibody" in the present specification and claims can be interchanged throughout to describe the determination of antibody concentration.
In the Anderson et al. application, it is pointed out that the peak value of the rate signal is double-valued in that a single peak value corresponds to two widely different antigen concentration values. On a plot of peak rate vs. antigen concentration, the peak rate increases from zero to a maximum and then decreases from the maximum with further increases in antigen concentration. On the ascending limb of the curve, at lower antigen concentrations, the reaction is in an antibody excess condition. On the descending limb, at higher antigen concentrations, the reaction is in antigen excess.
The measuring range of the nephelometer for measuring antigen concentrations is preferably on the ascending limb of the curve in the antibody excess region. Consequently, if it is determined that a measured peak rate value in fact corresponds to a higher antigen concentration on the descending limb in antigen excess, it is necessary to dilute and remeasure the sample one or more times until a peak rate measurement in antibody excess is obtained. Once an acceptable peak rate measurement in antibody excess is derived from the diluted sample, the corresponding antigen concentration value is scaled upward by the prior dilution factor to determine the actual antigen concentration of the original sample. Consequently, before a peak rate measurement can be accepted, it must be determined whether the reaction for which such peak rate was measured proceeded in antigen excess (requiring sample dilution and remeasurement) or in antibody excess (acceptable).
Several papers have appeared which deal with the nephelometric assay of antigen-antibody reactions and which address the problems in determining the antigen or antibody excess condition of such reactions. These references include: (1) Savory, J., Buffone, G. J., and Reich, R., Kinetics of the IgG-anti-IgG reaction as evaluated by conventional and stopped-flow nephelometry. Clin. Chem. 20, 1071 (1974); (2) Buffone, G. J., Savory, J., and Cross, R. E., Use of a laser-equipped centrifugal analyzer for kinetic measurement of serum IgG. Clin. Chem. 20, 1320 (1974); (3) Buffone, G. J., Savory, J., Cross, R. E., and Hammond, J. E., Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. I. Methodology. Clin. Chem. 21, 1731 (1975); (4) Buffone, G. J., Savory, J., and Hermans, J., Evaluation of kinetic light scattering as an approach to the measurement of specific proteins with the centrifugal analyzer. II. Theoretical considerations.
Clin. Chem. 21, 1735 (1975); and (5) Tiffany, T. O., Parella, J. M., Johnson, W. F., and Burtis, C. A., Specific protein analysis by light-scatter measurement with a miniature centrifugal fast analyzer. Clin. Chem. 20, 1055 (1974).
In references (1) and (2) the authors discuss a two-point, semi-kinetic method for measuring specific proteins by deriving the average rate of change of scatter between two fixed times. They recognize that scatter intensity rises more rapidly, in comparison with the end value it approaches, in antigen excess than in antibody excess. However, no method is suggested for utilizing such behavior for making an antigen or antibody excess determination. In fact, several of the same authors in subsequently published references (3) and (4) state "Consideration of later time intervals with the use of both PBS and PEG-PBS have not demonstrated any unique kinetic characteristics on which differentiation of either antigen or antibody excess samples could be based" (reference 3), and "Though probably more easily performed than the equilibrium method, the kinetic procedure cannot directly detect antigen excess at the present time" (reference 4). Consequently, while these authors recognize several fundamental properties of antigen-antibody reactions, they do not teach any kinetic methods for making antigen or antibody excess determinations.
The authors of reference (5) studied both the kinetic and the equilibrium measurement of antigen-antibody reactions and found better precision with equilibrium measurements. Again, as in references (1)-(4), no kinetic methods are disclosed in reference (5) for making antigen or antibody excess determinations. However, the authors do disclose a method of determining antigen excess for equilibrium measurements by measuring a change in equilibrium light scatter intensity caused by the post-addition of a small quantity of antibody into the reaction cell after the primary antigen-antibody reaction has reached equilibrium. If the primary reaction proceeded in an antigen excess condition, and additional antibody is injected into the reaction cell containing the equilibrated reaction components, the excess antigen reacts with the injected antibody and produces a significant change in scatter intensity. On the other hand, if the primary reaction proceeded in antibody excess, subsequent injection of the additional antibody produces an insignificant response. Consequently, the uniquely different responses upon post-addition of antibody provide an indication of the antigen or antibody excess condition of the primary reaction.
Unfortunately, while determination of antigen or antibody excess by the post-addition of reactant into the primary reaction is a reliable technique, it is time consuming to perform. In this regard, reference (5) employs the post-addition step only with equilibrium measurements. Consequently, a time delay is introduced while waiting for the primary reaction to reach equilibrium before the post-addition step. Post-addition delays are even more critical in nephelometers which analyze samples one at a time, as opposed to the simultaneous analysis in the centrifugal analyzer of reference (5), particularly where post-addition is performed on every sample. It should be noted that reference (5) is silent on any technique for determining antigen excess in kinetic measurements, whether by post-addition or by analysis of the kinetic data itself.
The aforementioned Anderson et al. application, on the other hand, sets forth several kinetic methods for determining antigen excess. In one method, the peak rate value and the elapsed time from the start of a reaction to occurrence of the peak rate are graphed as a function of increasing antigen concentration for a fixed antibody concentration. By an appropriate coordinate transformation technique described in the application, a plot is derived which establishes a single valued function, derived from the peak rate and the time thereto, distinguishing antigen excess from antibody excess. In a second method, the rate signal (which is the first derivative of the nephelometric signal) is differentiated to generate the second derivative of the nephelometric signal. The elapsed time from the start of the reaction to the occurrence of the peak of the rate signal is determined together with the time difference between the peak value of the rate signal and the peak value of the second derivative signal. A ratio is established of the elapsed time to the peak rate divided by the time difference between the peak values of the first and second derivative signals, and this ratio was found to distinguish antigen excess from antibody excess.
Both of the above mentioned Anderson et al. methods have the advantage of providing information during the normal course of a rate nephelometric analysis as to whether a sample is in antigen or antibody excess. While exhibiting such advantage, the methods appear to possess several drawbacks which reduce their current desirability for use in commercial instrumentation. For example, the working range for which the methods are optimized is somewhat restricted if one considers the various antigens and antibodies which a commercial instrument should measure. Moreover, the methods appear to be sensitive to noise and artifacts associated with injection transients.
In view of the foregoing, it would be desirable to derive a method for determining antigen or antibody excess in a kinetic environment which retains the reliability of the post-addition method but which minimizes the time delays heretofore associated therewith. The present invention fulfills these needs.